SHAPE Society

1. 1
STABILIZATION OFSTABILIZATION OF
VULNERABLE PLAQUESVULNERABLE PLAQUES
using
Ultra Super Paramagnetic Iron Oxide
and
Ferromagnetic Hyperthermia

2. 2
Vulnerable AtheroscleroticVulnerable Atherosclerotic
PlaquePlaque
Large lipid core
Thin Fibrous Cap
Macrophage Infiltration (inflammation)

3. 3
Vulnerable PlaqueVulnerable Plaque

4. 4
Factors Responsible forFactors Responsible for
Plaque InstabilityPlaque Instability
Increased macrophage infiltration
SMC apoptosis
Collagen degradation
Oxidized LDL
Plaque angiogenesis
Infection (CP, HSV, CMV)
Systemic inflammation / infections
(influenza)

5. 5
Factors Responsible forFactors Responsible for
Plaque StabilityPlaque Stability
Reduced Inflammation (Macrophage
Apoptosis?)
SMC Proliferation
Collagen Production
Lipid Lowering (lipid core)

6. 6
Methods of Stabilizing /Methods of Stabilizing /
Treatment of PlaquesTreatment of Plaques
Lipid lowering
Stent Placement
Anti inflammatory
Photodynamic therapy
Gene therapy
Beta blockers
ACE inhibitors
Antioxidants
Estrogen therapy
Antibiotics

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Photodynamic TherapyPhotodynamic Therapy
PDT is based on the use of light-sensitive
compounds,photo sensitizers (PS), which can be
activated by light of a specificwavelength that is
matched to the absorption characteristic of the
particular PS. Photo activation in the presence of
tissue oxygenleads to the formation of
cytotoxic-reactive oxygen species suchas singlet
oxygen. Photo activation of PS thereforemay
result in the damage of cellular components,
eventually leadingto cell death. (Levy, 1994).

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The rationale forusing PDT in
atherosclerosis is based on the observation
thatvarious PS, such as porphyrins,
phthalocyanines, and texaphyrins
accumulate in the atherosclerotic plaque as
compared with theadjacent normal vessel
wall. Localintra-arterial application of light
of the appropriate wavelengthto the
atherosclerotic vessel therefore may reduce
the narrowingof thevessel.

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Substances Used AsSubstances Used As
PhotosensitizersPhotosensitizers
porphyrins,
Phthalocyanines(2.6 times more in plaques after 4 hours)
Gadolinium texaphyrin
Lutetium texaphyrin
Tetracyclin
Pheophorbide
Mono-L-aspartyl chlorin e 6

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Texaphyrins in PDTTexaphyrins in PDT
( Lutetium Texaphyrins)( Lutetium Texaphyrins)
 These are aromatic and highly colored compounds
related to porphyrins.
 Lutetium texaphyrin is a second generation PS.
 It possesses a low energy maximum in the far red
portion of the visible spectrum.
 This provides for activation of these compounds
by light that is capable of penetration through
tissue and blood.
 It is retained selectively in the macrophages in the
atherosclerotic plaque in spite of rapid clearance
from blood.

11. 11
……ContinuedContinued
It can be used in photo angioplasty and
reduction of atherosclerotic plaque.
The efficacy of Lutetium Texaphyrin has
been demonstrated in animal models
It is in phase I clinical trial for coronary
artery procedure and in phase II for
peripheral arteries.

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Left: Pretreatment angiogram of the femoral artery of a patientLeft: Pretreatment angiogram of the femoral artery of a patient
showing the severe narrowing of this vessel caused byshowing the severe narrowing of this vessel caused by
atherosclerosis. Right: One month after a single treatment withatherosclerosis. Right: One month after a single treatment with
Lu-Tex-based ANTRINLu-Tex-based ANTRIN(TM)(TM)
photosensitizer-based angioplasty,photosensitizer-based angioplasty,
shows a 50% increase in the diameter of the arterial opening andshows a 50% increase in the diameter of the arterial opening and
increased blood flow. Reproduced with permission.increased blood flow. Reproduced with permission. 98 Pharmacyclics Inc.98 Pharmacyclics Inc.

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Biodistribution of LutetiumBiodistribution of Lutetium
TexaphyrinTexaphyrin (10mg/kg)(10mg/kg)
Time Plasmaµg/g Muscleµg/g Tumorµg/g
3 hours 5.07 0.60 5.25
5 hours 4.72 0.46 1.96
12 hours 3.91 0.41 0.48
24 hours 2.79 0.40 0.18

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Uptake of Porphyrins inUptake of Porphyrins in
arteries and plaquesarteries and plaques
Hsiang (1993) found that porphyrins are
taken up by human arteries in in-vitro
studies.
In in-vivo studies in miniswine these are
taken up by the plaques and normal arteries
in a ratio of 1.7-3.5 with a dose of
2.0mg/kg.

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Disadvantages of PDTDisadvantages of PDT
Coagulation necrosis, cell cytotoxicity
Burning plaque
Skin photosensitivity and severe skin burns
upon exposure to sun light
 Even LT is not highly plaque specific.

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Selective Targeting of PSSelective Targeting of PS
Selective targeting of PS to atherosclerotic
plaque may reduce its possible side effects, such
as skin hypersensitivity to sunlight
Photo activation of cells loaded with Ox LDL-
AlPc(aluminum phthalocyanine chloride)
complexes resulted in selective cell death that
correlated with the time of illumination. The
cytotoxic effect of OxLDL-AlPc on leukemic
murine macrophages is concentration- and
incubation time-dependent.[Vries 1999]

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Effects of Gentle HeatingEffects of Gentle Heating
Anti-Proliferative Effect of Heat
Anti-inflammatory Effects of Heat
Other Effects of Heat
Mitra Rajabi:Mitra Rajabi:
Mitra Rajabi:Mitra Rajabi:

18. 18
Anti-Proliferative Effect ofAnti-Proliferative Effect of
HeatHeat
Heat as well as other stressors (such as
withdrawal of growth factors, lack of oxygen
or glucose) can induce apoptosis, in a wide
variety of cell types.

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AntiAnti--inflammatory Effects ofinflammatory Effects of
HeatHeat
 Reduced expression of pro inflammatory
cytokines: TNF-α, IL-1 and IL-6 and many others
cytokines that are important mediators of
inflammation.
 Temperatures between 40°- 45 °C or chemical
inducers of heat shock proteins cause inhibition of
both TNF alpha and IL-1 expression.
 Inducing selective macrophage apoptosis.

20. 20
Effects of Heat onEffects of Heat on
CollagenCollagen(cap strengthening)(cap strengthening)
 Heat can increase expression of TGF-β 1 and
VGEF which are well known for their effects on
collagen and matrix production.
 Low grade heat therapy has been shown to help
the process of wound healing by increasing
collagen production in several studies. Heated
wounds showed increased collagen fibers which
were denser, thicker, better arranged and more
continuous, and the mean tensile strength of them
was significantly greater than control group.

21. 21
Effects of Heat on LipidEffects of Heat on Lipid
It has been shown that rise in temperature to
41ºC causes partial melting of the lipid
crystals in rabbit atherosclerotic plaque
At 45ºC most of the crystals melt.
Small, Arterosclerosis 1988

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Effect of Heat on MMP ActivityEffect of Heat on MMP Activity
Heat shock at 42°C preferentially
suppresses the production and expression of
MT1-MMP and thereby inhibits pro MMP-
2 (gelatinase) activation, events which
subsequently inhibits tumor invasion
Sato, 1999

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Disadvantages of Non-selectiveDisadvantages of Non-selective
HyperthermiaHyperthermia
Partial damage of the endothelial cell
membrane (at 54ºC for 5-10 minutes)
[Gabbiani,1984]
Swelling of the endothelial cell nuclei
(at43ºC for 12 minutes) [Emami, 1984]
Sticking of the WBC’s to the vessel walls
when heated, [Emami, 1984]

24. 24
Methods of InterstitialMethods of Interstitial
HyperthermiaHyperthermia
Radiofrequency
Microwave
Infrared Laser
Focused Ultra Sound
Extra corporeal heating of blood.
Whole Body Hyperthermia

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Invasive
 Catheters
 Ferromagnetic
Hyperthermia
 Surgical implantation
of NeedleThermo seed
 Extra corporeal
heating of blood.
Non Invasive.
 Inductive HT

26. 26
RF & Ferromagnetic HyperthermiaRF & Ferromagnetic Hyperthermia
Because the layers of tissues within the
human body have different water content
and dielectric properties, deep pattern of
exogenous energy absorption is highly non-
uniform. To make hyperthermia clinically
useful and feasible, it is essential that the
heat delivery techniques be able to provide
a sufficient heat dose within the whole site
of interest while leaving surrounding
normal tissue unaffected.

27. 27
Use of Ferromagnetic Micro ParticlesUse of Ferromagnetic Micro Particles
for Interstitial / Intracellularfor Interstitial / Intracellular
HyperthermiaHyperthermia
Heat production by metal devices implanted
directly into the tissues
Thermoseeds absorb energy from an
externally applied EM field in a non contact
manner.
Ferromagnetic microparticles and colloidal
magnetic particles act like thermoseeds but
in submicron and intracellualr level.

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Ferromagnetic particles act like self-
contained heaters for which no cable
connections are needed.
They dissipate energy by various kinds of
energy losses (eddy currents, hysteresis and
residual loss) and cause heat on exposure to
external alternating magnetic field.

29. 29
Amount of heat produced by
ferromagnetic particles is a
function of frequency (HZ) and
power (W) of currents created by
RF generator.
It varies from gentle heating (1 °
-2 ° C rise) to aggressive heating
and melting tissues (50°-70 ° C
rise )

30. 30
Ex Vivo StudiesEx Vivo Studies
1989-94 Dextran Magnetite Complex was
used to generate heat by exposing them to
AC magnetic field.
In 1994 Mitsumori compared heat
generation in DM/Lipiodal emulsion and
DM/Degradable starch micro sphere
emulsions.

31. 31

32. 32
…….continued.continued
1999-Shinkai, used agar containing
magnetite particles and heated them with
MHz-RF capacitative device. It resulted in
an increase in the temperature of the agar by
9ºC at 5minutes and by 24ºC at 9 minutes.
2000-Hilger, heated magnetite particles
placed in cow muscle tissue with 400 kHz
RF. Temperature elevation of up to 87ºC was
observed at a distance of 15mm from the
magnetite deposition area.

33. 33

34. 34
In Vivo Studies Using IronIn Vivo Studies Using Iron
Oxide ParticlesOxide Particles
1981-Gordon used Iron particles with
diameter of less than 1µ in rats and rabbits
and exposed them to external high
frequency electromagnetic field for 8- 10
minutes.
The treated animals showed in a marked
decrease in the lipid accumulation and
intimal hyperplasia

35. 35
Submicron colloidal carbon iron particles could be observed immediatelySubmicron colloidal carbon iron particles could be observed immediately
under the endothelium 5 hours after the administration, in mesentericunder the endothelium 5 hours after the administration, in mesenteric
artery, in a ratartery, in a rat

36. 36
……ContinuedContinued
1994- Mitsumori injected DM containing
embolic material to renal arteries of rabbits
and selectively heated the embolized kidney
by exposure to AC magnetic field.
The temperature of the kidney was raised
12.7º±3.1 during the initial 10 minutes.

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Iron Particles in the KidneyIron Particles in the Kidney

38. 38
……continuedcontinued
1999-Shinkai, injected CMC-Magnetite
(carboxy methyl complex) into the femur of
pigs. 8 MHz-RF was used to heat the
magnetite complex.
The temperature in the CMC-Mag complex
was increased to 43ºC after 7 minutes.

39. 39

40. 40
……continuedcontinued
1998 Merkle and associates, injected 6 male
NZ normal rabbits with ferumoxide and
applied RF field for 8 minutes.
Coagulative lesions were produced in the
liver of the rabbits which were detected by
MR imaging.

41. 41
Nonenhanced T1-
weighted SE image
(480/26 with three
signals acquired)
allows better
visualization of the
hypointense RF-
induced lesion
(arrows) [Merkle et al 1999-
Radiology]

42. 42
SPIO, USPIOSPIO, USPIO
Magnetic resonance imaging contrast
medium with a central core of iron oxide
generally coated by a polysaccharide layer
Shortening MR relaxation time
Phagocytosed and accumulated in cells with
phagocytic activity

43. 43
SPIO in Macrophages

44. 44
SPIOs AND USPIOsSPIOs AND USPIOs
These biocompatible particles are injectable
and nontoxic agents that have the ability to
dissipate the energy of radio frequency
electro magnetic field in the form of heat.
Can be useful in the site / cell specific
hyperthermia treatment.

45. 45
In Vivo Hyperthermia UsingIn Vivo Hyperthermia Using
CMIOCMIO(Colloidal Magnetic Iron Oxide)(Colloidal Magnetic Iron Oxide)
Dr Chan and associates evaluated the effect
of RF hyperthermia with CMIO injected
mice.
Mice were implanted with three types of
human lung carcinoma cells.
Tumors were exposed to RF field at 0.85
MHz for 6-10minutes.

46. 46
……continuedcontinued
Body vs tumor temperature differential of
6.5º-8.5ºC was reached.
Dr. Chan’s studies demonstrated the ability
to achieve clinically significant, site specific
heating of the tumors.

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 Uptake of Ferromagnetic particles by
macrophages in the plaque.
 Heating of the Ferromagnetic particles with low
frequency magnetic field.
 Increasing the temperature of the particles in the
macrophages to within the febrile range (38º-
43ºC)
 Apoptosis of the macrophages in the plaques
leading to decreasing inflammation and increasing
stability of the plaque.
SPIO Heating of VulnerableSPIO Heating of Vulnerable
PlaquesPlaques

48. 48
Our preliminary findings confirmed selective
accumulation of ferromagnetic particles in
the atherosclerotic plaque, RES system of
the liver and spleen as well as lymph nodes.

49. 49
Apo-e Mice 1 and 3 DaysApo-e Mice 1 and 3 Days
After Injection of FeridexAfter Injection of Feridex
Pearl’s Staining
Day1Day3

50. 50
HypothesesHypotheses
Radiofrequency heating of USPIO injected
animals for a short period of time (15-20
minutes at 42°-43°C) triggers apoptosis in
macrophages containing these particles and
therefore helps to stabilize the plaque by
reducing inflammation.
We also seek other beneficial effects of gentle
heating on collagen production as seen in wound
healing process, which may further help to
stabilize vulnerable plaque by cap strengthening.

51. 51
Study DesignStudy Design
Non-Invasive
Using inductive RF heating
Invasive
Using Intravascular cooled tip RF
catheter

52. 52
Using Intravascular catheter for applying
radiofrequency
For Non-Invasively heating the plaque we
need to improve the bio-distribution of
SPIOs to make them more plaque specific
so that hyperthermia can be induced non-
invasively

53. 53
Non-invasive Arm of theNon-invasive Arm of the
StudyStudy
15 with RF H ypertherm ia 5 without RF H ypertherm ia
20m ice U SPIO injection
5 with RF H ypertherm ia 5 without RF H ypertherm ia
10 withoutinjectionType title here
30 M ice
All animals sacrificed & then samples will be
send for Pathologic evaluation for apoptosis
5 Days Later
12hours later

54. 54
Invasive Arm of the StudyInvasive Arm of the Study
5 rab b it w ith R F 5 ra b b it w ith o u t R F
1 0 ra b b it w ith U S P IO in je ctio n
5 ra b b its w ith R F 5 ra bb its w ith o u t R F
1 0 ra b b it w ith o u t in je ctio n
2 0 W H H L ra b b it
After 5 days
All animals sacrificed & then samples will be send for
Pathologic evaluation for apoptosis
After 12 hours

55. 55

56. 56
Future PlanFuture Plan
Targeting of vulnerable plaque
Ox-LDL
– USPIO tagged with FC receptor for MDA
epitope of Ox-LDL
Adhesion Molecules
-VCAM / ICAM
- P-Selectin
- CD39

57. 57

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